1. Field of the Invention
The invention relates to nucleic acid based kits and methods for determining the presence or absence in a sample of agricultural pathogens.
2. Description of the Related Art
Agriculture is a major sector of the U.S. economy, accounting for more than 13% of the gross domestic product, and employing more than 15% of the U.S. population. Cattle and dairy farmers alone earn between $50 billion and $54 billion a year through meat and milk sales, and roughly $50 billion is raised every year through farm-related exports. Overall livestock sales in 2001 were in excess of $108 billion.
An agro-terrorism attack in the U.S. could cause devastating economic consequences, not only for the affected agribusinesses but also for allied industries and services, disrupting food supplies, trade, and tourism. Moreover, because of the structure of American agribusiness (e.g., highly concentrated herds with frequent movement, suboptimal animal tracking systems, minimal farm security/surveillance), agricultural and food industries are quite vulnerable to such an attack.
The agricultural community currently views an introduction of foot-and-mouth disease (FMD) into the United States as one of their greatest concerns. FMD is a severe, highly communicable viral disease of cattle, other ruminants, and swine, FMD is endemic to many countries in the world, and the virus is easy to obtain. Because FMD does not pose a direct threat to human health, there is no need for elaborate containment procedures or personal protective equipment while handling or preparing the virus. Recent estimates associated with the 2001 FMD outbreak in the United Kingdom place economic losses at greater than $30 billion (U.S.).
The Animal and Plant Health Inspection Service (APHIS), a branch of the U.S. Department of Agriculture (USDA), is charged with protecting the nation's livestock and poultry from the introduction of foreign animal diseases and for coordinating the response to an agricultural disease outbreak. The current system for detecting a foreign animal disease (FAD) such as FMD generally involves the following components: (1) observations by veterinary practitioners and livestock owners, who likely will be the first to suspect and report a FAD case; (2) investigation of suspect cases and submission of samples to USDA/APHIS at the Plum Island Animal Disease Center (PIADC); and (3) diagnostic work-up of tissues at the Plum Island Foreign Animal Diagnostic Disease Laboratory (FADDL). Currently, all testing for FMD is done (by law) at FADDL on Plum Island, which averages about 300 investigations per year. During a major outbreak, demand could rise to 100 investigations per week. The number of required diagnostic tests would far exceed current analysis capacity, and authorities would have to resort to subjective clinical observations to determine if herds must be destroyed.
A critical problem facing the USDA/APHIS and state agriculture departments in combating an outbreak of FMD is the lack of rapid, validated diagnostic assays for detection and identification of FMD. The need for improved diagnostics and surveillance programs to better enable the United Sates to detect and respond to foreign animal diseases (FADs) has been highlighted in several reports (e.g. National Association of State Departments of Agriculture and Research Foundation, the General Accounting Office, and the National Research Council) as well as Homeland Security Presidential Directive-9 (HSPD-9). Additionally, recent outbreaks of FMD in South America and the United Kingdom have heightened concerns about the ability of existing US surveillance systems to rapidly detect a FMD incursion early in the course of an outbreak and then provide the required diagnostic surge capacity needed for an outbreak response and the recovery of disease free status.
At PIADC, laboratory methods currently used to detect FADs include agar gel immunodiffusion assays, enzyme-linked immunosorbent assays (ELISA), serum neutralization assays, virus isolation in tissue culture, direct fluorescent antibody tests, electron microscopy, and animal inoculation. These methods are generally time-consuming and labor-intensive. Rapid polymerase chain reaction (PCR) assays have been used on a limited basis and a few rapid diagnostic tests are currently undergoing validation, but these tests are not yet available. (Callahan, J. D.; Brown, F.; Csorio, F. A.; Sur, J. H.; Kramer, E.; Long, G. W.; Lubroth, J.; Ellis. S. J.; Shoulars, K. S.; Gaffney, K. L.; Rock, D. L.; Nelson, W. M. “Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus.” J. Am. Vet. Med. Assn., 220 (11) 1636-1642, Jun. 1, 2002.)
APHIS also lacks rapid, validated diagnostic assays that can differentiate FMD from the many “look-alike” diseases indigenous to the animal agricultural industries (e.g., bovine viral diarrhea virus, contagious ecthyma, bluetongue virus). These domestic diseases induce signs that are similar to FMD: drooling, blisters, or lameness. In the absence of an FMD outbreak, these look-alike diseases can instill a sense of complacency on the part of practitioners and producers, who may have seen similar signs before and thus, believe these signs are the result of common enzootic diseases. When a practitioner notifies a regulatory agency of a suspicious disease, animal samples are sent to PIADC for FAD diagnosis only, not for diagnosis of look-alike domestic diseases. Consequently, without the ability to offer a timely diagnosis for the look-alike diseases that are indigenous, practitioners and producers might disregard important disease signs or develop a disinterest in becoming involved in an FAD investigation of their animals. In an actual FMD outbreak, however, the tendency is to err on the side of over-diagnosis. In the 2001 United Kingdom FMD outbreak, field diagnoses based on clinical observations resulted in a large number of false positive diagnoses (estimates are as high as 70-80%) and unnecessary slaughter of herds (4.3 million animals slaughtered), with only 2023 laboratory-confirmed cases of FMD.
Traditional approaches to DNA signature development started with the hypothesis that a particular gene was vital to the organism's virulence, host range, or other factors that might be considered “unique”. Suitable primers and probe were designed for the detection system of choice, with or without computational screening (via BLAST or equivalent) for uniqueness. The resulting assay would then be tested with the available strain(s) and success declared if the targets were detected, but the assay didn't detect whatever near-neighbors were tested. This approach would sometimes yield good results, but failures occurred due to inadequate strain panel coverage and cross reactions with genetic near neighbors and complex environmental samples.